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European Journal of Medicinal Chemistry Nov 2023A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium...
A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) in an in vitro assay were identified. SAR analysis showed that the most active compounds, which included a phenyl group bearing fluoro substituent(s) at C2, a methoxy function at C3, and a benzyl-heteroatom moiety at C6, exhibited in vitro MIC values generally around 0.63-1.26 μM against Mtb and Mm. However, these compounds were inactive against Mtb in vivo (mice), and investigations revealed very short metabolic half-lives (<10 min) when incubated with mouse liver microsomes. Multiple observations of side products produced from oxidative cleavage of the imidazole moiety during the chemical synthesis work suggested that this is a likely metabolic pathway leading to the lack of observed activity in vivo.
Topics: Animals; Mice; Mycobacterium tuberculosis; Antitubercular Agents; Mycobacterium marinum; Pyridazines; Microbial Sensitivity Tests
PubMed: 37524009
DOI: 10.1016/j.ejmech.2023.115637 -
Mutagenesis Mar 2017Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous...
Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis.
Topics: Amino Acid Sequence; Bacterial Proteins; Cloning, Molecular; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligases; DNA, Bacterial; Escherichia coli; Ku Autoantigen; Mycobacterium marinum; Mycobacterium tuberculosis; Plasmids; Sequence Alignment
PubMed: 27613236
DOI: 10.1093/mutage/gew042 -
Frontiers in Microbiology 2018is a close relative of that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. Sliding motility correlates with biofilm...
is a close relative of that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. Sliding motility correlates with biofilm formation and virulence in most bacteria. In this study, we used a sliding motility assay to screen 2,304 transposon mutants of NTUH-M6885 and identified five transposon mutants with decreased sliding motility. Transposons that interrupted the type VII secretion system (T7SS) ESX-1-related genes, (), (), and (), were present in 3 mutants. We performed reverse-transcription polymerase chain reaction to verify genes from to , which were found to belong to a single transcriptional unit. Deletion mutants of , , (), and () displayed significant attenuation regarding sliding motility and biofilm formation. NTUH-M6885 possesses a functional ESX-1 secretion system. However, deletion of or resulted in slightly decreased secretion of EsxB (which is also known as CFP-10). Thus, the ESX-1 secretion system mediates sliding motility and is crucial for biofilm formation. These data provide new insight into biofilm formation.
PubMed: 29899738
DOI: 10.3389/fmicb.2018.01160 -
Infection and Drug Resistance 2023Culture of is very time-consuming, taking several weeks to produce positive results. Seeking rapid and sensitive diagnostic methods for diagnosis can greatly improve...
PURPOSE
Culture of is very time-consuming, taking several weeks to produce positive results. Seeking rapid and sensitive diagnostic methods for diagnosis can greatly improve patient treatment. Our study aimed to compare the rapid diagnostic abilities of polymerase chain reaction (PCR), nested PCR and loop mediated isothermal amplification (LAMP) of detecting in skin samples from patients with infection.
METHODS
A total of 6 strains and 6 skin samples with definite diagnosis of infection were included in the study. We optimized LAMP performance for detection of genomic DNA and confirmed the specificity of the primers. Then, the sensitivity of the LAMP and nested PCR assays were assessed by strains and clinical samples.
RESULTS
Nested PCR was 10-fold more sensitive than the LAMP assay by serial dilution of DNA. PCR positive samples were all positive by LAMP detection of 6 clinical strains. Out of 6 clinical skin specimens confirmed as infection, 0 (0%), 3 (50%), 3 (50%), and 4 (66.6%) were positive by PCR, nested PCR, LAMP and culture. The LAMP shared the same sensitivity than nested PCR in strains and clinical samples, but it was easy to perform and faster than nested PCR assay.
CONCLUSION
Compared with conventional PCR, LAMP and nested PCR are more sensitive and have a higher detection rate of in clinical skin specimens. The LAMP assay proved to be more suitable for rapid diagnosis of infection in a shorter time, especially in resource-limited settings.
PubMed: 36969943
DOI: 10.2147/IDR.S404929 -
The Journal of Steroid Biochemistry and... Dec 2023The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as...
The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.
Topics: Mycobacterium marinum; Oxidation-Reduction; Cytochrome P-450 Enzyme System; Steroids; Sterols; Mycobacterium tuberculosis
PubMed: 37793577
DOI: 10.1016/j.jsbmb.2023.106406 -
Frontiers in Cellular and Infection... 2021Genome scale mutagenesis identifies many genes required for mycobacterial infectivity and survival, but their contributions and mechanisms of action within the host are...
Genome scale mutagenesis identifies many genes required for mycobacterial infectivity and survival, but their contributions and mechanisms of action within the host are poorly understood. Using CRISPR interference, we created a knockdown of gene in (), which reduced the resistance to acid medium. To further explore the function of PPE31, the mutant strain was generated in and (), respectively. Macrophages infected with the mutant strain caused a reduced inflammatory mediator expressions. In addition, macrophages infected with Δ had decreased host cell death dependent on JNK signaling. Consistent with these results, deletion of from increased the sensitivity to acid medium and reduced cell death in macrophages. Furthermore, we demonstrate that both mutants from and resulted in reduced survival in macrophages, and the survivability of was deceased in zebrafish due to loss of . Our findings confirm that PPE31 as a virulence associated factor that modulates innate immune responses to mycobacterial infection.
Topics: Animals; Cell Death; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Mycobacterium tuberculosis; Virulence; Zebrafish
PubMed: 33928042
DOI: 10.3389/fcimb.2021.629836 -
Immunity Oct 2016In tuberculosis, some macrophages in granulomas assume an epitheloid appearance. Using the Mycobacterium marinum-zebrafish model, Cronan et al. (2016) now show that... (Review)
Review
In tuberculosis, some macrophages in granulomas assume an epitheloid appearance. Using the Mycobacterium marinum-zebrafish model, Cronan et al. (2016) now show that granuloma macrophages undergo reprograming events involving E-cadherin-dependent formation of epithelial-like cell-cell junctions. Interference with the function of E-cadherin in macrophages disorganized the granulomas and protected the fish, introducing new ideas and questions about macrophage function and granulomatous diseases.
Topics: Animals; Cadherins; Granuloma; Humans; Intercellular Junctions; Macrophages; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum
PubMed: 27760333
DOI: 10.1016/j.immuni.2016.10.002 -
MBio Apr 2023The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium...
The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6CMHCII monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6CMHCII monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.
Topics: Animals; Mice; Neutrophils; Bacterial Proteins; Type VII Secretion Systems; Mycobacterium tuberculosis; Mycobacterium marinum; Inflammation; Cell Differentiation
PubMed: 37017530
DOI: 10.1128/mbio.02764-22 -
PLoS Pathogens Jan 2017During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium...
During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium tuberculosis. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the Dictyostelium discoideum/Mycobacterium marinum infection model we showed that M. marinum accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. Dictyostelium knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, M. marinum alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the dgat1&2 double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During Dictyostelium infection, ILI formation in M. marinum is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy.
Topics: Chromatography, Thin Layer; Dictyostelium; Fluorescent Antibody Technique; Host-Pathogen Interactions; Inclusion Bodies; Microscopy, Electron, Transmission; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Phospholipids; Triglycerides
PubMed: 28103313
DOI: 10.1371/journal.ppat.1006095 -
Cureus Nov 2022is a non-tuberculous mycobacteria present in natural and non-chlorinated bodies of water. It is a known fish pathogen but can also cause human disease. It usually...
is a non-tuberculous mycobacteria present in natural and non-chlorinated bodies of water. It is a known fish pathogen but can also cause human disease. It usually causes cutaneous lesions but in rare cases may originate more invasive diseases with the involvement of deep structures. We describe three cases of patients with cutaneous infection by evaluated in a tertiary care center, two with confirmed infection and one with a presumptive diagnosis based on clinical and epidemiological features. A brief bibliographic review of infections is then presented to support the theme. We aim to alert one to the difficulties in establishing the correct diagnosis of this infection, emphasize the importance of a high degree of suspicion for its identification, and review the therapeutic management options.
PubMed: 36579262
DOI: 10.7759/cureus.31787